High molecular weight esthetic compositions

ABSTRACT

Described are high molecular weight glycosaminoglycan (GAG) hydrogel compositions comprising GAGs covalently crosslinked with a carbohydrate crosslinker, and methods of making the high molecular weight GAG hydrogel compositions. Further described are methods of using the high molecular weight glycosaminoglycan (GAG) hydrogel compositions for reparative or plastic surgery, esthetic dermatology, facial contouring, body contouring, and gingival augmentation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/108,729, filed Dec. 1, 2020, which claims the benefit of priority toU.S. application Ser. No. 62/942,624, filed Dec. 2, 2019, which areherein incorporated by reference in their entireties.

FIELD

The present disclosure relates to the field of high molecular weightesthetic compositions such as hydrogels containing crosslinkedpolysaccharides, and the use of such hydrogels in medical and/orcosmetic applications such as implants for subcutaneous or intradermalinjection, which may be used in humans in reparative or plastic surgeryand in esthetic dermatology. More specifically, the present disclosureis concerned with hydrogels comprising crosslinked high molecular weightglycosaminoglycans (GAGs), particularly crosslinked hyaluronic acid,chondroitin, or chondroitin sulfate.

BACKGROUND

Hydrogels are widely used in medicine—prepared by chemical crosslinkingpolymers to form large polymeric networks. While both monomeric andminimally polymerized polysaccharides both absorb water to the point ofsaturation, polysaccharides dissolve at the point of saturation whilehydrogels comprising the same polysaccharides, albeit crosslinked, cantypically absorb water without dissolving, resulting in a swelling ofthe hydrogel.

All glycosaminoglycans (GAGs) are negatively charged long linearheteropolysaccharides that have a capacity to absorb large amounts ofwater. Hyaluronic acid, chondroitin, and chondroitin sulfate arewell-known biocompatible GAGs utilized in medical and cosmeticapplications. One of the most widely used biocompatible polymers formedical use is hyaluronic acid, and derivatives thereof. Modifyinghyaluronic acid molecules through crosslinking and other means isnecessary to improve the duration of hyaluronic acid in vivo, such ascrosslinking hyaluronic acid to form a hyaluronic acid hydrogel.

Producing hydrogels from high molecular weight GAGs, such as hyaluronicacid results in a suitable filler for multiple types of medical orcosmetic applications; however, the hydrogel may degrade or hydrolyzeduring storage or during degrading conditions such as heat sterilizationor accelerated stability studies. Methods of increasing the stability ofthe hydrogels include increasing the number of crosslinks in thehydrogels, but increasing the number of crosslinks of high molecularweight GAGs can produce hydrogels that are stable under degradingconditions but results in a phase separation of the hydrogel. Thispresents problems to then diluting hydrogels without obtaining phaseseparation.

The aim of the present disclosure is to overcome the problems associatedwith preparing hydrogels from high molecular weight GAGs that exhibitincreased stability and are able to maintain the integrity of thehydrogel during degradation conditions, such as heat sterilization,while maintaining the ability to dilute hydrogels to desired GAGconcentrations for applications such as filling syringes with hydrogels.

SUMMARY OF THE DISCLOSURE

The present disclosure is generally drawn to methods of producing ahydrogel from crosslinked high molecular weight glycosaminoglycans(GAGs) capable of maintaining the structural integrity under conditionsthat would otherwise hydrolyze the hydrogel or result in a phaseseparation of the hydrogel. The present disclosure is further drawn tohydrogel compositions produced by the methods.

In some aspects, the disclosure is generally drawn to a method ofpreparing a hydrogel comprising crosslinked glycosaminoglycan (GAG)molecules, said method comprising (a) crosslinking a GAG having amolecular weight of at least 1.5 MDa with a crosslinker, wherein theconcentration of GAG is between 2% to 10% (w/w) and the molar ratio ofthe crosslinker to GAG is less than or equal to 2%, to obtain aglycosaminoglycan hydrogel crosslinked by amide bonds.

In some aspects, if the GAG concentration in (a) is between 1% to 4.5%(w/w), then the concentration of crosslinker is between 0.8 to 2 mol %per GAG disaccharide; and if the GAG concentration in (a) is between4.6% to 5.9% (w/w), then the concentration of crosslinker is between 0.5to 0.8 mol % per GAG disaccharide; and if the GAG concentration in (a)is between 6% to 12% (w/w), then the concentration of crosslinker isbetween 0.3 to 0.5 mol % per GAG disaccharide.

In some aspects, the crosslinker in (a) is a di- or multinucleophilefunctional crosslinker. In some aspects, the di- or multinucleophilefunctional crosslinker is an aliphatic or aromatic diamino derivative, apeptide or a peptide sequence. In some aspects, the di- ormultinucleophile functional crosslinker comprises a spacer groupselected from the group consisting of di-, tri-, tetra-, andoligosaccharides. In some aspects, the di- or multinucleophilefunctional crosslinker is diaminotrehalose (DATH).

In some aspects, the crosslinking of (a) comprises: a1) providing orobtaining a solution of glycosaminoglycan (GAG) molecules; a2)activating carboxyl groups on the glycosaminoglycan molecules with acoupling agent to form activated glycosaminoglycan molecules; and a3)crosslinking the activated glycosaminoglycan (GAG) molecules via theiractivated carboxyl groups using a di- or multinucleophile functionalcrosslinker to obtain a glycosaminoglycan hydrogel crosslinked by amidebonds. In some aspects, the coupling agent used in a2) is atriazine-based coupling agent, such as4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM).

In some aspects, the crosslinking in (a) is performed at a pH of5.0-9.0, preferably at a pH of 6.0-8.0. In some aspects, the methodsfurther comprise the formulating the crosslinked hydrogel obtained from(a) to a final glycosaminoglycan (GAG) concentration of 10-30 mg/mL. Insome aspects, the methods further comprise (b) sterilizing thecrosslinked hydrogel obtained from (a).

In some aspects, the glycosaminoglycan (GAG) is hyaluronic acid (HA). Insome aspects, the GAG in (a) has a molecular weight of 2.0-10 MDa,preferably 2.5-3.5 MDa. In some aspects, the concentration of GAG isbetween 3-5% (w/w). In some aspects, the molar ratio of crosslinker toGAG is between 0.9-1.1%. In some aspects, the GAG is hyaluronic acid(HA) having a molecular weight of 2.5-3.5 MDa and the crosslinker isdiaminotrehalose (DATH), and further wherein the concentration of HA isbetween 3-5% (w/w) and the molar ratio of DATH to HA is between0.9-1.1%.

In some aspects, the disclosure is generally drawn to a hydrogel productobtained by any one of the methods described herein. In some aspects,the disclosure is generally drawn to a hydrogel product comprisingglycosaminoglycan (GAG) molecules as the swellable polymer, wherein theglycosaminoglycan molecules are crosslinked by amide bonds and whereinthe apparent molecular weight (Mwapp) of the crosslinkedglycosaminoglycan molecules is above 1.0 MDa; and wherein thethermostability of the swellable polymer (NormGelC) is above 80% after24 hours.

In some aspects, the glycosaminoglycan molecules are covalentlycrosslinked via crosslinks comprising a spacer group selected from thegroup consisting of di-, tri-, tetra-, and oligosaccharides. In someaspects, the spacer group is trehalose. In some aspects, theglycosaminoglycan (GAG) is hyaluronic acid (HA). In some aspects, thehydrogel product is sterilized.

According to any one or more of the previous aspects, the GAG does nothave a molecular weight of less than 1.5 MDa, or optionally, less than1.4 MDa, 1.3 MDa, 1.2 MDa, 1.1 MDa, 1.0 MDa, 0.9 MDa, 0.8 MDa, or 0.7MDa.

According to any one or more of the above aspects, the hydrogel is notsubjected to a post-crosslinking degradation of the glycosaminoglycan.According to any one or more of the above aspects, the hydrogel issubject to ambient degradation post-crosslinking; however, the hydrogeldoes not exhibit a C_(min) value below that of C_(final)/2. According toany one or more of the above aspects, the hydrogel exhibits a C_(min)value greater than C_(final)/2 of the hydrogel.

In some aspects, the (a) and (b) are performed stepwise from (a) to (b).In some aspects, al), a2), and a3) are performed stepwise from al) toa2) to a3). In some aspects, (a) and (b) are not performed stepwise from(a) to (b). In some aspects, al), a2), and a3) are not performedstepwise from al) to a2) to a3).

In some aspects, the disclosure is generally drawn to a method ofcosmetically treating skin, which comprises administering to the skin ahydrogel product according to any one of the hydrogel compositionsdescribed herein.

The following detailed description is exemplary and explanatory, and isintended to provide further explanation of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the normalized gel content (NormGelC (%)) for four gelsamples that exhibit various HA (MW) and HA crosslinking over a periodof approximately 70 hours at 90° C.

FIG. 2 depicts the gel content (GelC %) corresponding to samples 1 and 2in Table 2 after the gels were incubated at 90° C. for 24 or 48 hours.

FIG. 3 depicts the gel content (GelC %) corresponding to samples 3 and 4in Table 2 after the gels were incubated at 90° C. for 24 or 48 hours.

FIG. 4 depicts the gel content (GelC %) corresponding to samples 5 and 6in Table 2 after the gels were incubated at 90° C. for 24 or 48 hours.

FIG. 5 depicts the gel content (GelC %) corresponding to samples 7 and 8in Table 2 after the gels were incubated at 90° C. for 24 or 48 hours.

DETAILED DESCRIPTION OF THE DISCLOSURE I. Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” may refer to one or more of that entity, i.e. canrefer to plural referents. As such, the terms “a” or “an”, “one or more”and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

Reference throughout this specification to “one embodiment”, “anembodiment”, “one aspect”, or “an aspect” means that a particularfeature, structure or characteristic described in connection with theembodiment is included in at least one embodiment of the presentdisclosure. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics can be combined inany suitable manner in one or more embodiments.

As used herein, the terms “about” or “approximately” when preceding anumerical value indicates the value plus or minus a range of 10% of thevalue.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

As used herein, a “control” is an alternative sample used in anexperiment for comparison purpose. A control can be “positive” or“negative.” A “control sample” or “reference sample” as used herein,refers to a sample or reference that acts as a control for comparison toan experimental sample. For example, an experimental sample comprisescompound A, B, and C in a vial, and the control may be the same type ofsample treated identically to the experimental sample, but lacking oneor more of compounds A, B, or C.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount which results in the prevention of one or more outcomes,or an increase in one more outcomes.

As used herein, the terms “individual”, “patient”, or “subject” can bean individual organism, a vertebrate, a mammal, or a human. In apreferred aspect, the individual, patient, or subject is a human.

As used herein, the phrase “soft tissue” refers to tissues that connect,support, or surround other structures and organs of the body. Softtissue includes muscles, fibrous tissues, and fat.

As used herein, the phrase “soft tissue augmentation” refers to any typeof volume augmentation of soft tissues, including, but not limited tofacial contouring (e.g., more pronounced cheeks, chin, or lips),correction of concave deformities (e.g., post-traumatic orHIV-associated lipoatrophy), and correction of deep age-related facialfolds. Thus, soft tissue augmentation may be used for cosmetic purposesor for medical purposes, such as those following trauma or degenerativedisease. Soft tissue augmentation further refers to dermal filling, bodycontouring, and gingival filling.

As used herein, the phrase “non-animal origin” refers to a source thatexcludes animals, but includes sources such as yeast, bacteria, orsynthetic.

As used herein, the term “bioresorbable” refers to a degradation eventor events—bioresorbable substances may dissolve, may be phagocytized, ormay simply degrade over a period of time such that the substances arecleared from the body, organ, tissue, location, or cell over a period oftime. The substances or degradation products thereof may be metabolized,incorporated into other molecules or compounds, or excreted.

As used herein, the term “aseptic” refers to something that is free orfreed from pathogenic microorganisms.

As used herein, the term “sterile” refers to something that is free ofliving organisms, generally free of living microorganisms.

As used herein, the term “injectable” refers to the ability to inject acomposition of the present disclosure through a needle.

As used herein, the terms “MW” or “Mw” refer to the mass averagemolecular mass.

As used herein, the term “MWapp” refers to apparent MW, which is asimulated value for the molecular weight of GAGs in hydrogels.

As used herein, the term “SwF” refers to the swelling factor analysis insaline, which is the volume of saline for a 1 gram gel that has swelledto its maximum—usually represented in mL/g).

As used herein, “gel content” or “GelC” refer to the percentage in theproportion of the total HA that is bound in gel form—further describedas the amount of HA in a sample that does not pass through a 0.22micrometer filter. The GelC is calculated from the amount of HA that iscollected in the filtrate and is reported as the percentage of the totalamount of HA in the gel sample.

As used herein, “SwD” refers to the swelling degree, which is theinverted concentration of gel-form GAG in a gel that is fully swollen in0.9% saline, i.e., the volume or mass of a fully swollen gel that can beformed per gram of dry crosslinked GAG. The SwD generally describes themaximal liquid-absorbing (0.9% saline) capability of the product. SwD ispreferably expressed in g/g, mL/g, or as a dimensionless number.

${SwD} = \frac{{mass}\left( {{fully}{swollen}{gel}} \right)}{{mass}\left( {{gel} - {form}} \right.{GAG}{in}{fully}{swollen}{gel}}$

The SwD may also be expressed as

${SwD} = \frac{\lbrack{GAG}\rbrack*{Gel}{}C}{SwF}$

As used herein, “CrR_(DATH)” refers to the effective crosslinking ratiothat was analyzed with LC-SEC-MS, more specifically defined as

${CrR} = \frac{{mol}{crosslinked}{crosslinker}{with}{amide}{bonds}}{{mol}{linked}{crosslinker}{with}{amide}{bonds}}$

A CrR of 1.0 indicates that all of the crosslinker has crosslinked.

As used herein, “C_(min)” is the minimum theoretical GAGconcentration—the concentration of gel-form GAGs in a gel that is fullyswollen in 0.9% saline, typically expressed in mg/g or mg/mL.

The C_(min) ⁻¹=SwD

As used herein, “C_(final)” is the intended concentration of the GAG inthe final hydrogel product. In some aspects, C_(final) is greater than2×C_(min).

The present technology is not to be limited in terms of the particularaspects described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the present technology. It is to beunderstood that this present technology is not limited to particularmethods, reagents, compounds compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

II. Hydrogels and Methods of Making Hydrogels

Fillers such as dermal fillers have been used to repair, restore oraugment hard or soft tissue contour defects of the body due to aging,injury, or acquired or congenital deformities of the face, body andinternal organs. Fillers may be natural or synthetic substances that areused to reduce wrinkles and/or fine lines, restore lost volume, hydratethe skin, soften nasolabial folds, augment and contour lips, improvescars (depressed, hypertrophic and keloid scars), strengthen weakenedvocal cords, and provide other soft tissue improvements. Substances thathave been utilized include fat, paraffin, human collagen, bovinecollagen, silicone, hyaluronic acids, lactic acids, and glycolic acids.In 1981, a new era in soft tissue fillers emerged with the FDA approvalof bovine collagen. Since then, many soft tissue fillers have emerged.The dramatic increase in the number of current and investigationalfillers has been fueled by many factors including improvements inbiotechnology and an emphasis on cosmetic appearance in society. Withthe introduction of newer fillers, there has been an ongoing need toevaluate their risk/benefit profiles and define their limitations inorder to maximize patient cosmetic outcomes and safety. Commonfiller/hydrogel compositions include GAGs such as hyaluronic acid.

Methods of producing GAG hydrogels are disclosed in PCT publicationnumbers WO2017/114867, WO2017/114861, WO2017/114864, and WO2017/114865;US Pregrant Publication Numbers US20190023812A1, US20190016830A1,US20190023855A1, and US20070066816A1; and U.S. Pat. Nos. 8,858,999,6,831,172, 8,887,243, and, 6,703,444.

A common route for crosslinking hyaluronic acid is using a diglycidylether, e.g. 1,4-butanediol diglycidyl ether (BDDE). As an alternative,amide coupling using a di- or multiamine functional crosslinker togetherwith a coupling agent is an attractive route for preparing crosslinkedhyaluronic acid molecules useful for hydrogel products. For example, theuse of 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride (DMTMM) for activation of carboxylate and subsequentcondensation with a diamino structure, e.g. diaminotrehalose (DATH) hasshown to be an efficient method to produce hydrogels composed ofcrosslinked hyaluronic acid with minor degradation of the biopolymer.

In some aspects, crosslinking is performed via crosslinkers comprising aspacer group selected from the group consisting of di-, tri-, tetra-,and oligosaccharides. This provides a hydrogel product based entirely oncarbohydrate structures or derivatives thereof, which minimizes thedisturbance of the crosslinking on the native properties of the GAGsutilized in producing the hydrogel.

In some aspects, the crosslinker itself contributes to maintained orincreased properties of the hydrogel, for example when crosslinking witha structure that correlates to hyaluronic acid (e.g., diamino hyaluronicacid tetrasaccharide) or when crosslinking with a structure with highwater retention properties (e.g., trehalose).

In some aspects, the GAG is a sulfated or non-sulfated GAG such ashyaluronan, chondroitin sulphate, heparan sulphate, heparosan, heparin,dermatan sulphate and keratan sulphate. In some aspects, the GAG ishyaluronic acid, chondroitin or chondroitin sulfate. In one aspect, theGAG is hyaluronic acid. In some aspects, the GAG is a native GAG. Insome aspects, the GAG is a naturally occurring GAG. In some aspects, theGAG is used in its native state (i.e., the chemical structure of the GAGhas not been altered or modified by addition of functional groups or thelike). Using the GAG in its native state is preferred because this willafford a crosslinked structure more closely resembling the naturalmolecules, which conserves the native properties and effects of the GAGitself, and can minimize the immune response when the crosslinked GAG isintroduced into the body.

In some aspects, the GAGs are covalently crosslinked. In some aspects,the covalently crosslinked GAG molecules consist of, or essentiallyconsist of carbohydrate type structures or derivatives thereof. In someaspects, the crosslinked GAGs or hydrogels are free of, or essentiallyfree of synthetic non-carbohydrate structures or linkers. This can beachieved by using a GAG in its native state together with a crosslinkerwhich comprises, consists of, or essentially consist of carbohydratetype structures or derivatives thereof. In some aspects, functionalgroups of the crosslinker are covalently bound directly to carboxylgroups of the GAG. In some aspects, the crosslinks of the covalentlycrosslinked GAGs comprise, consist of, or essentially consist of di-,tri-, tetra-, and oligosaccharide spacer groups.

In some aspects, the crosslinked GAG comprises crosslinks between theGAG molecule chains, which creates a continuous network of GAG moleculesheld together by covalent crosslinks.

In some aspects, the crosslinked GAGs form a gel orhydrogel—water-insoluble, but substantially dilute crosslinked system ofGAGs when subject to liquid, typically an aqueous liquid.

In some aspects, the process of preparing a hydrogel product comprisingcrosslinked glycosaminoglycan molecules, comprises, consists of, orconsists essentially of: (a) providing a solution of glycosaminoglycanmolecules; (b) activating carboxyl groups on the glycosaminoglycanmolecules with a coupling agent to form activated, glycosaminoglycanmolecules; and (c) crosslinking the activated glycosaminoglycanmolecules via their activated carboxyl groups using a di- ormultinucleophile functional crosslinker comprising a spacer groupselected from the group consisting of di-, tri-, tetra-, andoligosaccharides to obtain crosslinked glycosaminoglycan molecules.

In some aspects, the GAGs are crosslinked by covalent bonds, such asamide bonds, typically using an activating agent for the carboxyl groupson the GAG molecule backbone and a di- or multinucleophile functionalcrosslinker comprising a spacer group selected from the group consistingof di-, tri-, tetra-, and oligosaccharides. In some aspects,crosslinking of the GAGs can be achieved by mild and efficient routesresulting in high yields with minimal degradation of the GAG molecules.

In some aspects, the di- or multinucleophile functional crosslinkercontains a spacer group selected from the group consisting of di-, tri-,tetra-, and oligosaccharides, which remains in the crosslinks betweenthe GAG molecules. In some aspects, the di- or multinucleophilefunctional di-, tri-, tetra-, and oligo-saccharides comprise at leasttwo nucleophile functional groups attached thereto. In some aspects, theat least two nucleophile functional groups are separated by the spacergroup selected from the group consisting of di-, tri-, tetra-, andoligosaccharides.

In some aspects, the di- or multinucleophile functional crosslinkercomprises two or more functional groups capable of reacting withfunctional carboxyl groups of the GAG, resulting in the formation ofcovalent bonds, such as amide bonds. In some aspects, the nucleophilefunctional groups are capable of reacting with carboxyl groups on theglycosaminoglycan molecule to form amide bonds. In some aspects, thenucleophile functional groups of the di-, tri-, tetra-, andoligosaccharides are selected from the group consisting of primaryamine, hydrazine, hydrazide, carbazate, semi-carbazide,thiosemicarbazide, thiocarbazate and aminoxy. In some aspects, the di-or multinucleophile functional di-, tri-, tetra-, and oligo-saccharidesmay be derived from nucleophile functional polysaccharides, such aschitobiose derived from chitin. In some aspects, the di- ormultinucleophile functional di-, tri-, tetra-, and oligo-saccharides mayalso be di-, tri-, tetra-, and oligo- saccharides which have beenmodified by introduction of two or more nucleophile functional groups.

In some aspects, the di- or multinucleophile functional crosslinkerinclude homo- or heterobifunctional primary amines, hydrazines,hydrazides, carbazates, semi-carbazides, thiosemicarbazides,thiocarbazates and aminoxy.

In some aspects, the crosslinker is selected from the group consistingof diamino hyaluronic acid tetrasaccharide, diamino hyaluronic acidhexasaccharide, diamino trehalose (DATH), diamino lactose, diaminomaltose, diamino sucrose, diamino chitobiose, chitobiose, or diaminoraffinose.

In some aspects, the activation step and the crosslinking step occursimultaneously. In some aspects, the activation step occurs prior to andseparately from the crosslinking step.

In some aspects, a step subsequent to crosslinking comprises providingparticles of the crosslinked GAG molecule, having an average size in therange of 0.01-5 mm, preferably 0.1-0.8 mm.

In some aspects, the particles are between 20 to 800 μm in size. In someaspects, the particles are between about 100 to about 500 μm in size. Insome aspect, this size may be length, diameter, or width. In general,this refers to diameter. In some aspects, the particles are between 20to 800 μm, between 20 to 700 μm, between 20 to 600 μm, between 20 to 500μm, between 20 to 400 between 20 to 300 between 20 to 200 between 100 to800 μm, between 100 to 700 μm, or between 100 to 300 μm in size.

In some aspects, the coupling agent is a peptide coupling reagent. Insome aspects the coupling reagent is selected from4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT). A preferredtriazine-based peptide coupling reagent is DMTMM. Other preferredpeptide coupling reagent are carbodiimide coupling reagents, preferablyN-(3-dimethylanninopropyl)-N′-ethylcarbodiinnide (EDC) combined withN-hydroxysuccinimide (NHS).

In some aspects, crosslinking of the activated GAG molecules occurs viatheir carboxyl groups using a crosslinker. In some aspects, thecrosslinker is a di- or multinucleophile functional crosslinkercomprising a spacer group selected from the group consisting of di-,tri-, tetra-, and oligosaccharides. In some aspects, the crosslinkerconnects the GAG chains to each other via carboxyl groups on the GAGbackbone. In some aspects, the spacer group may be a hyaluronic acidtetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose,maltose, sucrose, cellobiose or raffinose residue. By the term “residue”is meant here that the structure of the compound is similar but notidentical to the patent compounds hyaluronic acid tetrasaccharide,hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose,cellobiose or raffinose respectively. The structure of the residue maydiffer from the structure of the parent compound in that it has beenprovided with two or more nucleofile functional groups and optionallycovalently linked via said nucleophile functional groups carboxyl groupson the GAG backbone.

According to a related aspect, the present invention also provides useof the hydrogel product as a medicament, such as in the treatment ofsoft tissue disorders. There is provided a method of treating a patientsuffering from a soft tissue disorder by administering to the patient atherapeutically effective amount of the hydrogel product. There is alsoprovided a method of providing corrective or aesthetic treatment to apatient by administering to the patient a therapeutically effectiveamount of the hydrogel product.

In some aspects, the hydrogel contains mostly liquid by weight and cancontain 90-99.9%, water, but it behaves like a solid due to athree-dimensional crosslinked GAG molecule network within the liquid.Due to its significant liquid content, the hydrogel is structurallyflexible and similar to natural tissue, which makes it very useful as ascaffold in tissue engineering and for tissue augmentation. It is alsouseful for treatment of soft tissue disorder and for corrective oresthetic treatment. In some aspects, the hydrogel is used as aninjectable formulation.

The methods disclosed herein are methods of using the injectablecompositions for reparative or plastic surgery, esthetic dermatology,facial contouring, body contouring, and gingival augmentation. In someaspects, the compositions are freeze-dried or lyophilized. In someaspects, the compositions comprise a hydrogel comprising an aqueoussolution.

In some aspects, a crosslinked glycosaminoglycan product, such ashyaluronic acid product, is produced from high molecular weight (HMW)glycosaminoglycan using amide crosslinking.

In some aspects, a crosslinked hyaluronic acid product from HMWhyaluronic acid using DATH/DMTMM chemistry is formulated to create asuitable GAG concentration for use (C_(final) of 10-45 mg/mL). In someaspects, the GAG is a hyaluronic acid. In some aspects, the suitable GAGconcentration is for dermatological use, dental use, medical use, orreconstructive surgical use.

In some aspects, the suitable GAG concentration is 10 to 50 mg/mL, 10 to45 mg/mL, 10 to 40 mg/mL, 10 to 35 mg/mL, 10 to 30 mg/mL, 10 to 25mg/mL, 10 to 20 mg/mL, 10 to 15 mg/mL, 15 to 40 mg/mL, 15 to 40 mg/mL,15 to 35 mg/mL, 15 to 30 mg/mL, 15 to 25 mg/mL, 15 to 20 mg/mL, 20 to 50mg/mL, 20 to 45 mg/mL, 20 to 40 mg/mL, 20 to 35 mg/mL, 20 to 30 mg/mL,20 to 25 mg/mL, 25 to 50 mg/mL, 25 to 45 mg/mL, 25 to 40 mg/mL, 25 to 35mg/mL, 25 to 30 mg/mL, 30 to 50 mg/mL, 30 to 45 mg/mL, 30 to 40 mg/mL,30 to 35 mg/mL, 35 to 50 mg/mL, 35 to 45 mg/mL, 35 to 40 mg/mL, 40 to 50mg/mL, or 40 to 45 mg/mL.

In some aspects, the suitable GAG concentration is about 10 to about 50mg/mL, about 10 to about 45 mg/mL, about 10 to about 40 mg/mL, about 10to about 35 mg/mL, about 10 to about 30 mg/mL, about 10 to about 25mg/mL, about 10 to about 20 mg/mL, about 10 to about 15 mg/mL, about 15to about 40 mg/mL, about 15 to about 40 mg/mL, about 15 to about 35mg/mL, about 15 to about 30 mg/mL, about 15 to about 25 mg/mL, about 15to about 20 mg/mL, about 20 to about 50 mg/mL, about 20 to about 45mg/mL, about 20 to about 40 mg/mL, about 20 to about 35 mg/mL, about 20to about 30 mg/mL, about 20 to about 25 mg/mL, about 25 to about 50mg/mL, about 25 to about 45 mg/mL, about 25 to about 40 mg/mL, about 25to about 35 mg/mL, about 25 to about 30 mg/mL, about 30 to about 50mg/mL, about 30 to about 45 mg/mL, about 30 to about 40 mg/mL, about 30to about 35 mg/mL, about 35 to about 50 mg/mL, about 35 to about 45mg/mL, about 35 to about 40 mg/mL, about 40 to about 50 mg/mL, or about40 to about 45 mg/mL.

In some aspects, hyaluronic acid encompasses all variants andcombinations of variants of hyaluronic acid, hyaluronate, orhyaluonan—of various chain lengths and charge states, as well as variouschemical modifications, including crosslinking.

In some aspects, hyaluronic acid encompasses the various hyaluronatesalts of hyaluronic acid with various counter ions, such as sodiumhyaluronate. In some aspects, various modifications of hyaluronic acidare also encompassed by recitation of hyaluronic acid, such asoxidation, e.g. oxidation of —CH₂OH groups to —CHO and/or —COOH;periodate oxidation of vicinal hydroxyl groups, which may be followed byreduction, e.g. reduction of —CHO to —CH₂OH or coupling with amines toform imines followed by reduction to secondary amines; sulphation;deamidation, which may be followed by deamination or amide formationwith new acids; esterification; crosslinking; substitutions with variouscompounds, e.g. using a crosslinking agent or a carbodiimide assistedcoupling; including coupling of different molecules, such as proteins,peptides and active drug components, to hyaluronic acid; anddeacetylation. In some aspects, hyaluronic acid may be further modifiedby isourea, hydrazide, bromocyan, monoepoxide, and monosulfonecouplings.

In some aspects, hyaluronic acid may be obtained from various sources ofanimal and non-animal origin. In some aspects, sources of non-animalorigin include yeast or bacteria. In some aspects, the molecular weightof a single hyaluronic acid molecule is typically in the range of 0.1 to10 mDa, but other molecular weights are possible.

In some aspects, the present disclosure is drawn to at least the partialdeacetylation of a biopolymer/hydrogel comprising acetyl groups,comprising: a) providing a biopolymer comprising acetyl groups; b)reacting the biopolymer comprising acetyl groups with hydroxylamine or asalt thereof at a temperature of 100° C. or less for 2 to 200 hours toform an at least partially deacetylated biopolymer, and c) recoveringthe at least partially deacetylated biopolymer. In some aspects, thehydrogel is prepared from HMW GAGs through the methods described herein.

In some aspects, the present disclosure is drawn to a method ofpreparing a hydrogel product comprising crosslinked GAGs, the methodcomprising: a) providing a GAG crosslinked by amide bonds, wherein thecrosslinked GAGs comprise residual amine groups, and b) acylatingresidual amine groups of the crosslinked GAGS provided in a) to formacylated crosslinked GAGs. In some aspects, the hydrogel is preparedfrom HMW GAGs through the methods described herein.

In some aspects, the present disclosure is drawn to a method ofpreparing a hydrogel product comprising crosslinked GAGs, the methodcomprising: a) providing a GAG crosslinked by aminde bonds, wherein thecrosslinked GAGs comprise ester crosslinks formed as byproducts duringthe amide crosslinking; and b) subjecting the crosslinked GAGs toalkaline treatment to hydrolyze ester crosslinks formed as byproductsduring the amide crosslinking. In some aspects, the hydrogel is preparedfrom HMW GAGs through the methods described herein.

When producing hydrogels from a high molecular weight (HMW)glycosaminoglycan, such as hyaluronic acid, with the DATH/DMTMM systemusing low DATH loading, a suitable gel for filler composition isinitially formed, but the hydrogel may hydrolyze upon storage or duringexposure to degrading conditions (e.g. heat sterilization, acceleratedstability studies). This indicates that a larger number of crosslinks isneeded to keep the gel intact and protect it against hydrolysis.However, increasing the amount of DATH/DMTMM—to increase the number ofcrosslinks in the gel—may lead to gels with high C_(min) that are phaseseparated at suitable GAG concentrations. In other words, when producinggels from HMW GAGs with enough crosslinks to make them stable duringautoclaving it isn't possible to dilute the gels to 20 mg/ml (10-45mg/ml) without obtaining phase separation. This will in turn addcomplexity to the process for e.g. filling the gel in syringes. In someaspects, the hydrogel is homogeneous. A homogeneous product is not phaseseparated. In some aspects, the hydrogel is formulated to a suitableconcentration for dermatological use (such as 10-45 mg/mL), but retainsthe capacity to swell in the presence of excess saline.

In some aspects, the method of producing a hydrogel does not result inphase separation of the hydrogel. In some aspects, a hydrogel producedor derived from the methods disclosed herein is not phase separated. Insome aspects, the method of diluting a hydrogel after heat sterilizationdoes not result in phase separation of the hydrogel.

In some aspects, the hydrogel is diluted in a PBS buffer. In someaspects, the hydrogel is diluted in a 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM,17 mM, 18 mM, 19 mM, or 20 mM phosphate buffer. In some aspects, thehydrogel is diluted in about 1 mM, about 2 mM, about 3 mM, about 4 mM,about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM,about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM phosphatebuffer. In some aspects, the hydrogel is diluted to between 1 mM to 20mM, 1 mM to 15 mM, 1 mM to 10 mM, 1 mM to 5 mM, 5 mM to 20 mM, 5 mM to15 mM, 5 mM to 10 mM, 10 mM to 20 mM, 10 mM to 15 mM, or 15 mM to 20 mMphosphate buffer. In some aspects, the hydrogel is diluted to betweenabout 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM toabout 10 mM, about 1 mM to about 5 mM, about 5 mM to about 20 mM, about5 mM to about 15 mM, about 5 mM to about 10 mM, about 10 mM to about 20mM, about 10 mM to about 15 mM, or about 15 mM to about 20 mM phosphatebuffer.

In some aspects, the hydrogel is diluted in a solution at a pH of about6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2,about 7.4, about 7.6, about 7.8, or about 8.0. In some aspects, thehydrogel is diluted in a solution at a pH of 6.0, 6.2, 6.4, 6.6, 6.8,7.0, 7.2, 7.4, 7.6, 7.8, or 8.0. In some aspects, the hydrogel isdiluted in a solution at a pH of between 6.0 to 8.0, between 6.0 to 7.0,between 7.0 and 8.0, between 6 and 7.5, between 7.0 and 7.5, or between6.5 and 7.5.

In some aspects, the HA concentration of the crosslinking reaction isabout 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%,about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, or about 20%. In some aspects, the HAconcentration of the crosslinking reaction is 1%, 1.5%, 2%, 2.5%, 3%,3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some aspects,the HA concentration of the crosslinking reaction is between 1% to 3%,2% to 5%, 3% to 5%, 4% to 5%, 2% to 3%, 2% to 4%, 3% to 5%, 3% to 4%, 1%to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 1% to 2%, 2% to 5%, 2% to 10%,2% to 15%, 2% to 20%, 5% to 20%, 5% to 15%, 5% to 10%, 10% to 20%, 10%to 15%, or 15% to 20%.

In some aspects, the HA concentration of the crosslinking reaction isnot greater than 10%. In some aspects, the HA concentration of thecrosslinking reaction is not greater than 9%. In some aspects, the HAconcentration of the crosslinking reaction is not greater than 8%. Insome aspects, the HA concentration of the crosslinking reaction is notgreater than 7%. In some aspects, the HA concentration of thecrosslinking reaction is not greater than 6%. In some aspects, the HAconcentration of the crosslinking reaction is not greater than 5%. Insome aspects, the HA concentration of the crosslinking reaction is notgreater than 4%. In some aspects, the HA concentration of thecrosslinking reaction is not greater than 3%.

In some aspects, the mol-% of DATH in the crosslinking reaction is 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%,1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2%. In some aspects, the mol-% ofDATH in the crosslinking reaction is about 0.1%, about 0.2%, about 0.3%,about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%,about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%,about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%. In someaspects, the mol-% of DATH in the crosslinking reaction is 0.1% to 2%,0.1% to 1.5%, 0.1% to 1%, 0.1% to 0.5%, 0.2% to 1%, 0.2% to 0.5%, 0.2%to 0.3%, 0.5% to 1%, 0.5% to 1.5%, 0.5% to 2%, 1% to 2%, or 1% to 1.5%.

In some aspects, the GAG has a molecular weight of above 700 kDa, 800kDa, 900 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2500 kDa, 3000kDa, 3500 kDa, 4000 kDa, 4500 kDa, 5000 kDa, 5500 kDa, 6000 kDa, 6500kDa, 7000 kDa, 7500 kDa, 8000 kDa, 8500 kDa, 9000 kDa, 9500 kDa, or10000 kDa.

In some aspects, the GAG has a molecular weight of above about 700 kDa,about 800 kDa, about 900 kDa, about 1000 kDa, about 1100 kDa, about 1200kDa, about 1300 kDa, about 1400 kDa, about 1500 kDa, about 1600 kDa,about 1700 kDa, about 1800 kDa, about 1900 kDa, about 2000 kDa, about2500 kDa, about 3000 kDa, about 3500 kDa, about 4000 kDa, about 4500kDa, about 5000 kDa, about 5500 kDa, about 6000 kDa, about 6500 kDa,about 7000 kDa, about 7500 kDa, about 8000 kDa, about 8500 kDa, about9000 kDa, about 9500 kDa, or about 10000 kDa.

In some aspects, the thermostability (NormGelC or GelC) of the hydrogelis at least 80% after 24 hours. In some aspects, the thermostability(NormGelC or GelC) of the hydrogel is at least 80% after 48 hours. Insome aspects, the thermostability (NormGelC or GelC) of the hydrogel isat least 80% after 24 hours at about 90° C. In some aspects, thethermostability (NormGelC or GelC) of the hydrogel is at least 80% after24 or 48 hours at a temperature of at least 70° C. In some aspects, thethermostability (NormGelC or GelC) of the hydrogel is at least 80% after24 or 48 hours at a temperature of at least 90° C.

In some aspects, the thermostability (NormGelC or GelC) of the hydrogelis at least 70%, 75%, 80%, 85%, 90%, or 95% after 24 hours or 48 hours.In some aspects, the thermostability (NormGelC or GelC) of the hydrogelis at least 70%, 75%, 80%, 85%, 90%, or 95% after 24 hours or 48 hoursat a temperature of at least 70° C. or at least 90° C.

In some aspects, the thermostability (NormGelC or GelC) of the hydrogelis at least about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% after 24 hours or 48 hours. In some aspects, thethermostability (NormGelC or GelC) of the hydrogel is at least 70%, 75%,80%, 85%, 90%, or 95% after 24 hours or 48 hours at a temperature of atleast 70° C. or at least 90° C.

In some aspects, the thermostability (NormGelC or GelC) of the hydrogeldecreases by less than 5%, 10%, 15%, 20%, 25%, or 30% after 24 hours or48 hours at a temperature of at least 70° C. or at least 90° C. In someaspects, the thermostability (NormGelC or GelC) of the hydrogeldecreases by less than about 5%, about 10%, about 15%, about 20%, about25%, or 30% after 24 hours or 48 hours at a temperature of at least 70°C. or at least 90° C.

In some aspects, the temperature at which the thermostability isdetermined is at least 70° C., 72° C., 74° C., 76° C., 78° C., 80° C.,82° C., 84° C., 86° C., 88° C., 90° C., 92° C., 94° C., 96° C., 98° C.,100° C., 102° C., 104° C., 106° C., 108° C., or 110° C. In some aspects,the temperature at which the thermostability is determined is at leastabout 70° C., about 72° C., about 74° C., about 76° C., about 78° C.,about 80° C., about 82° C., about 84° C., about 86° C., about 88° C.,about 90° C., about 92° C., about 94° C., about 96° C., about 98° C.,about 100° C., about 102° C., about 104° C., about 106° C., about 108°C., or about 110° C.

In some aspects, the period of time post-manufacture at which thethermostability is determined is at least 24 hours, 26 hours, 28 hours,30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72hours, 74 hours, 76 hours, 78 hours, or 80 hours. In some aspects, theperiod of time post-manufacture at which the thermostability isdetermined is at least about 24 hours, about 26 hours, about 28 hours,about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours,about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56hours, about 58 hours, about 60 hours, about 62 hours, about 64 hours,about 66 hours, about 68 hours, about 70 hours, about 72 hours, about 74hours, about 76 hours, about 78 hours, or about 80 hours.

In some aspects the composition is bioresorbable. In some aspects, thehydrogel is bioresorbable. In some aspects, the composition isbioresorbed within a period of about 1 year to about 3 years. In someaspects, the composition is bioresorbed within a period of 1 year to 3years. In some aspects, the hydrogel is bioresorbed within a period ofabout 1 year to about 3 years. In some aspects, the hydrogel isbioresorbed within a period of 1 year to 3 years.

In some aspects, the composition further comprises a local anesthetic.In some aspects, the composition comprises at least one localanesthetic. In some aspects the local anesthetic is an amide-type localanesthetic. In some aspects, the local anesthetic is an ester-type localanesthetic.

In some aspects, the local anesthetic is selected from the groupconsisting of: bupivacaine, butanilicaine, carticaine, cinchocaine(dibucaine), clibucaine, ethyl parapiperidinoacetylaminobenzoate,etidocaine, lignocaine (lidocaine), mepivacaine, oxethazaine,prilocaine, ropivacaine, tolycaine, trimecaine, vadocaine, articaine,levobupivacaine, amylocaine, cocaine, propanocaine, clormecaine,cyclomethycaine, proxymetacaine, amethocaine (tetracaine), benzocaine,butacaine, butoxycaine, butyl aminobenzoate, chloroprocaine,dimethocaine (larocaine), oxybuprocaine, piperocaine, parethoxycaine,procaine (novocaine), propoxycaine, and tricaine; or a combinationthereof.

In some aspects, the concentration of local anesthetic in thecomposition is between 1 to 5 mg/mL. In some aspects, the concentrationof local anesthetic in the composition is between about 1 to about 5mg/mL. In some aspects, the concentration of local anesthetic in thecomposition is between 2 to 4 mg/mL. In some aspects, the concentrationof local anesthetic in the composition is between about 2 to about 4mg/mL. In some aspects, the concentration of local anesthetic in thecomposition is 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 2.5 mg/mL, 3mg/mL, 3.5 mg/mL, 4 mg/mL, 4.5 mg/mL, or 5 mg/mL. In some aspects, theconcentration of local anesthetic in the composition is about 0.5 mg/mL,about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 2.5 mg/mL, about 3mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, or about 5mg/mL.

In some aspects, the composition is injectable. In some aspects, theinjectable composition is an injectable implant. In some aspects, thedisclosure is drawn to an injectable implant comprising any one of thecompositions disclosed herein. In some aspects, the injectable implantis for subdermal, intradermal, subcutaneous, intramuscular, submuscular,intragingival injection.

In some aspects, the disclosure is drawn to a pre-filled syringecomprising any one of the compositions disclosed herein. In someaspects, the disclosure is drawn to a pre-filled vial comprising any oneof the compositions disclosed herein.

In some aspects, a kit comprises a pre-filled syringe comprising any oneof the compositions disclosed herein. In some aspects, a kit comprises apre-filled vial comprising any one of the compositions disclosed herein,a syringe, and one or more hypodermic needles. In some cases the kitcomprises an antimicrobial composition for administering to the site ofinjection.

In some aspects, kits for use in practicing the methods described hereinare contemplated. In some aspects, kits comprise all solutions, buffers,compounds, vessels, and/or instructions sufficient for performing themethods described herein.

In some aspects, the composition further comprises sodium chloride. Insome aspects, the composition exhibits a sodium chloride concentrationof 0.9% w/v. In some aspects, the composition further comprises aphosphate buffer. In some aspects, the composition further comprises apharmaceutically acceptable carrier. In some aspects the compositionfurther comprises sodium chloride, a phosphate buffer, and apharmaceutically acceptable carrier.

In some aspects, the composition comprises one or more density enhancingagents. In some aspects, the density enhancing agents may be selectedfrom sorbitol, mannitol, and fructose.

In some aspects, the composition comprises a buffering agent. Abuffering agent is a chemical compound that is or compounds that areadded to a solution to allow that solution to resist changes in pH as aresult of either dilution or small additions of acids or bases.Effective buffer systems employ solutions which contain large andapproximately equal concentrations of a conjugate acid-base pair (orbuffering agents). A buffering agent employed herein may be any suchchemical compound(s) which is pharmaceutically acceptable, including butnot limited to salts (conjugates acids and/or bases) of phosphates andcitrates. In some aspects, the buffering agent comprises phosphatebuffered saline (PBS) or an alternative phosphate buffer.

In some aspects, the composition is aseptic. In some aspects, thecomposition is sterile. In some aspects, the composition is sterilizedvia filtration sterilization, heat sterilization, or irradiationsterilization. In some aspects, components of the composition aresterilized prior to mixing or forming the whole composition, thusresulting in a composition that comprises two or more components thatwere sterilized prior to forming the composition.

In some aspects, the GAG does not have a molecular weight of less than1.5 MDa. In some aspects, the GAG does not have a molecular weight ofless than 1.4 MDa. In some aspects, the GAG does not have a molecularweight of less than 1.3 MDa. In some aspects, the GAG does not have amolecular weight of less than 1.2 MDa. In some aspects, the GAG does nothave a molecular weight of less than 1.1 MDa. In some aspects, the GAGdoes not have a molecular weight of less than 1.0 MDa. In some aspects,the GAG does not have a molecular weight of less than 0.9 MDa. In someaspects, the GAG does not have a molecular weight of less than 0.8 MDa.In some aspects, the GAG does not have a molecular weight of less than0.7 MDa.

In some aspects, the hydrogel is not subjected to a post-crosslinkingdegradation of the glycosaminoglycan. In some aspects, the hydrogel issubject to ambient degradation post-crosslinking; however, the hydrogeldoes not exhibit a C_(min) value below that of C_(final)/2. In someaspects, the hydrogel exhibits a C_(min) value greater than C_(final)/2of the hydrogel.

Other aspects and preferred embodiments of the present invention will beevident from the following detailed disclosure of the invention and theappended claims.

II. Methods of Using the Hydrogels

In some aspects, the present disclosure comprises methods of performingreparative or esthetic dermatologic treatment. In some aspects, thereparative or esthetic dermatologic treatment comprises injecting asubject with a composition disclosed herein. In some aspects, theinjection is a subdermal, intradermal, subcutaneous, intramuscular,submuscular, or intragingival injection.

In some aspects, methods of the present disclosure are drawn tointragingival injection to fill the gums as a result of receding gums.In some aspects, methods are drawn to injection of the composition inone or more tissues of the oral cavity.

In some aspects, the injection is for dermal filling, body contouring,facial contouring, and gingival filling.

In some aspects, the injection of a composition disclosed herein is fordermal filling. In some aspects, methods of dermal filling includeinjection of the composition to fill skin cracks. In some aspects,methods of dermal filling include injection of the composition to fillfine lines in the face, neck, hands, feet, knees, and elbows. In someaspects, methods of dermal filling include injection of the compositionto fill fine wrinkles in the face, neck, hands, feet, knees, and elbows.In some aspects, methods of dermal filling include injection of thecomposition to fill fine lines in the face, neck, hands, feet, knees,and elbows.

In some aspects, methods of dermal filling include injection of thecomposition to fill scars. In some aspects, methods of dermal fillinginclude injection of the composition to fill depressed scars. In someaspects, methods of dermal filling include injection of the compositionto fill hypertrophic scars. In some aspects, methods of dermal fillinginclude injection of the composition to fill keloid scars.

In some aspects, methods of dermal filling include injection of thecomposition to restore and/or correct for signs of facial fat loss(lipoatrophy) in people with human immunodeficiency virus (HIV).

In some aspects, methods of dermal filling include injection of thecomposition to the backs of hands or the top of feet.

In some aspects, methods of dermal filling include injection of thecomposition to strengthen weakened vocal cords.

In some aspects, methods of dermal filling include injection of thecomposition to restore lost volume to a portion of the body as a resultof age, illness, or injury.

In some aspects, methods of facial contouring include injection of thecomposition to the face to modify the facial contour. In some aspects,methods of facial contouring include injection of the composition to thelips to augment the size and/or shape of the lips.

In some aspects, methods of facial contouring include injection of thecomposition to the face to increase facial symmetry. In some aspects,methods of facial contouring include injection of the composition tochange the shape of the face to an oval shape, round shape, squareshape, triangle shape, inverted triangle shape, rectangular shape, oroblong shape. In some aspects, methods of facial contouring includeinjection of the composition to increase the total width of the face. Insome aspects, methods of facial contouring include injection of thecomposition to increase the total length of the face.

In some aspects, methods of facial contouring include injection of thecomposition to the face to increase the forehead and/or cheekbone width.In some aspects, methods of facial contouring include injection of thecomposition to the face to increase the length of the jawline.

In some aspects, methods of facial contouring include injection of thecomposition to the face to change the size and/or shape of the chin. Insome aspects, methods of facial contouring include injection of thecomposition to the face to change the size and/or shape of the forehead.In some aspects, methods of facial contouring include injection of thecomposition to the face to change the size and/or shape of the cheeks.In some aspects, methods of facial contouring include injection of thecomposition to the face to change the size and/or shape of the brow.

In some aspects, methods of facial contouring include injection of thecomposition to the face to modify the appearance associated withretrognathia. In some aspects, methods of facial contouring includeinjection of the composition to the face to modify the appearanceassociated with prognathism.

In some aspects, methods of body contouring include injection of thecomposition to the body to modify the size and shape of various aspectsof the body. In some aspects, methods of body contouring includeinjection of the composition to the body to modify the size and shape ofaspects of the body to increase symmetry.

In some aspects, methods of body contouring include injection of thecomposition to the body to modify the size and shape of the breasts,buttocks, sacrum, groin, hips, abdomen, thorax, feet, legs, knees,popliteus, thighs, arms, hands, elbows, and/or antecubitis,

In some aspects, methods of body contouring include injection of thecomposition to the body to fill a concave deformity. In some aspects,the concave deformity is a result of age, illness, injury, orpredisposition. In some aspects, methods of body contouring includeinjection of the composition to the body to decrease the appearance ofcellulite.

EXAMPLES Example 1 High Molecular Weight Hydrogel Production

Example 1 provides the general process for making high molecular weightGAG hydrogels described herein.

Hyaluronic acid (HA) and solutions of diamino trehalose (DATH) and4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM) are mixed to crosslink. After the crosslinking process, the gelis divided and diluted to a set concentration. Thereafter, the gel isheated to approximately 70° C. for approximately 24 hours. The gel thenundergoes particle size reduction (PSR) and precipitation, and is thenwashed and dried to a powder. The gel powder is then mixed into asuitable buffer and the gel swells; the gel is then autoclaved.

TABLE 1 Hydrogel compositions corresponding to FIG. 1. ReactionConditions Gel Properties DATH/HA [HA] Mw SwF GelC C_(final) Sample (mol%) (%) (MDa) (mL/g) (%) CrR (mg/g) 1-1 0.9 3.5 1.0 5.2 87 ND 20 1-2 0.94 1.0 4.2 87 ND 20 1-3 0.9 3.5 2.1 4 96 ND 20 1-4 0.9 4 2.1 3.2 98 0.3119 ND = Not determined

The Gel Content was analyzed under heat degradation conditions at 90° C.and evaluated over the course of just over 70 hours (FIG. 1 ). The gelcontent values are normalized for ease of comparison, which is depictedas “NormGelC (%)” in the y-axis of FIG. 1 . The data indicates that thehigh molecular weight HA provides gels that maintain a higher degree ofgel content under heat degradation conditions as compared to the gelsmade from the lower molecular weight HA.

Example 2 Preparing and Evaluating High Molecular Weight (HMW) and LowMolecular Weight (LMW) hyaluronic acid (HA) Hydrogels General procedurefor crosslinking BMW and LMW HA

Stock solutions of each of DMTMM and the DATH crosslinker were freshlyprepared in water. A reaction solution was prepared by adding desiredvolumes of the DMTMM (mol % DATH×8.5) and DATH (see Table 1) stocksolutions, respectively, to water. The reaction solution was mixed anddirectly added to pre-weighted HA (0.4 MDa or 2.1 MDa) in a reactionvessel. The mixture was extensively mixed for 3 min and incubated. After24±2 h, the obtained material was pressed through a 1 mm steel mesh. Thematerial was homogenized for 24±2 h at 70° C. before the gel wassubjected to particle size reduction (PSR) using a 3×315 μm filter andthen precipitated by adding EtOH. The obtained powder was dried undervacuum overnight and reconstituted in 7 mM phosphate, 0.7% NaCl and 3mg/g Lido-HCl at neutral pH. The gel was filled in syringes andsubsequently autoclaved.

TABLE 2 Reactions conditions and obtained gel properties. ReactionConditions Gel Properties DATH/HA [HA] Mw SwF GelC C_(final) Sample (mol%) (%) (MDa) (mL/g) (%) CrR (mg/g) 2-1 1.5 2 0.4 10.6 44 0.17 20 2-2 1.52 2.1 6.2 91 0.20 20 2-3 0.3 10 0.4 5.4 74 0.55 40 2-4 0.3 10 2.1 2.7 950.50 40 2-5 0.5 6 0.4 5.9 75 0.38 25 2-6 0.5 6 2.1 2.9 94 0.37 25 2-70.9 4 0.4 4.9 83 0.32 20 2-8 0.9 4 2.1 3.2 98 0.28 20

The gels were incubated in sealed glass vials at 90° C. in a water bathfor 24 hours or 48 hours. At the given time, the samples were cooled toroom temperature and the gels were analyzed. See FIGS. 2-4

FIG. 2 depicts the gel content (GelC %) corresponding to samples 2-1 and2-2 in Table 2. FIG. 3 depicts the gel content (GelC %) corresponding tosamples 2-3 and 2-4 in Table 2. FIG. 4 depicts the gel content (GelC %)corresponding to samples 2-5 and 2-6 in Table 2. FIG. 5 depicts the gelcontent (GelC %) corresponding to samples 2-7 and 2-8 in Table 2.

General Procedure for Determining the MW_(app) in HMW Gels

Pre-weighted HA (2.1 MDa) was mixed with water in a reaction vessel. Themixture was extensively mixed for 3 min and incubated at ambienttemperature. After 24 hours, the solution was diluted with water,NaCl(s) was added (final concentration 0.9%), and the obtained materialwas incubated at 70° C. After incubation for 24 hours, the solution wasfilled in syringes. Syringes for sample 2-10 was subsequently autoclavedwhile the syringes for sample 2-9 were not. The Mw of the solutions weredetermined by SEC-MALLS.

TABLE 3 Reaction conditions and obtained gel properties. ReactionConditions DATH/ HA Properties (mol- [HA] Mw Mw_(app) Sample %) (%)(MDa) (MDa) Polydisp. Comment 2-9 2 4 2.1 2.2 2.2 Not autoclaved 2-10 04 2.1 1.4 2.1 Autoclaved

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the disclosure claimed. Thus, it should beunderstood that although the present disclosure has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the disclosure embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this disclosure.

The disclosure has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the methods. This includes thegeneric description of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. The presenttechnology is not to be limited in terms of the particular embodimentsdescribed in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the present technology. It is to beunderstood that this present technology is not limited to particularmethods, reagents, compounds compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

One skilled in the art readily appreciates that the present disclosureis well adapted to carry out the objects and obtain the ends andadvantages mentioned, as well as those inherent therein. Modificationstherein and other uses will occur to those skilled in the art. Thesemodifications are encompassed within the spirit of the disclosure andare defined by the scope of the claims, which set forth non-limitingembodiments of the disclosure.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed:
 1. A hydrogel product comprising a hyaluronic acidhydrogel crosslinked by amide bonds, obtained by a method comprising:crosslinking a hyaluronic acid (HA) having a molecular weight of 2.1 to10 MDa with a crosslinker, to obtain the hyaluronic acid hydrogelcrosslinked by amide bonds, wherein: the crosslinker is a di- ormultinucleophile functional crosslinker comprising a spacer groupselected from the group consisting of di-, tri-, tetra-, andoligosaccharides; the concentration of GAG is between 2% to 10% (w/w);and the molar ratio of the crosslinker to GAG is less than or equal to 2mol % per GAG disaccharide.
 2. The hydrogel product according to claim1, wherein the spacer group is trehalose.
 3. The hydrogel productaccording to claim 1, wherein the di- or multinucleophile functionalcrosslinker is diaminotrehalose (DATH).
 4. The hydrogel productaccording to claim 1, wherein the HA has a molecular weight of 2.1-3.5MDa.
 5. The hydrogel product according to claim 1, wherein: the HA has amolecular weight of 2.1-3.5 MDa; the crosslinker is diaminotrehalose(DATH); and during the crosslinking, the concentration of HA is between3-5% (w/w) and the molar ratio of DATH to HA is between 0.9-1.1 mol %per HA disaccharide.
 6. The hydrogel product according to claim 1,wherein the hydrogel product is essentially free of syntheticnon-carbohydrate structures or linkers.
 7. The hydrogel productaccording to claim 1, wherein the crosslinked HA molecules have anapparent molecular weight (Mwapp) above 1.0 MDa.
 8. The hydrogel productaccording to claim 1, wherein the crosslinked HA molecules have anapparent molecular weight (Mwapp) of 2.1 MDa or above.
 9. The hydrogelproduct according to claim 1, wherein the hydrogel product has a HAconcentration of 10-45 mg/mL.
 10. The hydrogel product according toclaim 1, wherein the crosslinking comprises: (1) providing a solution ofthe HA molecules; (2) activating carboxyl groups on the HA moleculeswith a coupling agent to form activated HA molecules; (3) crosslinkingthe activated HA molecules via their activated carboxyl groups using adi- or multinucleophile functional crosslinker to obtain a HA hydrogelcrosslinked by amide bonds.
 11. The hydrogel product according to claim1, wherein the concentration of the GAG is between 3-5% (w/w) during thecrosslinking.
 12. The hydrogel product according to claim 1, wherein themolar ratio of crosslinker to HA is between 0.9-1.1 mol % per GAGdisaccharide during the crosslinking.
 13. The hydrogel product accordingto claim 1, further comprising a local anesthetic.
 14. The hydrogelproduct according to claim 13, wherein the local anesthetic compriseslidocaine.
 15. A syringe comprising the composition according toclaim
 1. 16. A method of performing a reparative or estheticdermatologic treatment, the method comprising: injecting a subject withthe composition of claim
 1. 17. The method of claim 16, wherein theinjecting is performed as a subdermal, intradermal, subcutaneous,intramuscular, submuscular, or intragingival injection.
 18. The methodof claim 16, wherein the reparative or esthetic dermatologic treatmentcomprises dermal filling.
 19. The method of claim 18, wherein theinjection is administered to fill lines, fine lines, wrinkles, or skincracks.
 20. The method of claim 19, wherein the lines, fine lines,wrinkles, or skin cracks are located on the face, neck, hands, feet,knees, or elbows.